Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies possible targets of allele-specific immunity to clinical malaria.

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. Immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode possible targets of naturally acquired immunity that should be validated immunologically and further characterized for their potential as vaccine candidates.

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum.

BACKGROUND: The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points. METHODS: GC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates. RESULTS: GC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007-2008 and 2014-2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002. CONCLUSIONS: The GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Genetic diversity and drug resistance surveillance of Plasmodium falciparum for malaria elimination: is there an ideal tool for resource-limited sub-Saharan Africa?

The intensification of malaria control interventions has resulted in its global decline, but it remains a significant public health burden especially in sub-Saharan Africa (sSA). Knowledge on the parasite diversity, its transmission dynamics, mechanisms of adaptation to environmental and interventional pressures could help refine or develop new control and elimination strategies. Critical to this is the accurate assessment of the parasite's genetic diversity and monitoring of genetic markers of anti-malarial resistance across all susceptible populations. Such wide molecular surveillance will require selected tools and approaches from a variety of ever evolving advancements in technology and the changing epidemiology of malaria. The choice of an effective approach for specific endemic settings remains challenging, particularly for countries in sSA with limited access to advanced technologies. This article examines the current strategies and tools for Plasmodium falciparum genetic diversity typing and resistance monitoring and proposes how the different tools could be employed in resource-poor settings. Advanced approaches enabling targeted deep sequencing is valued as a sensitive method for assessing drug resistance and parasite diversity but remains out of the reach of most laboratories in sSA due to the high cost of development and maintenance. It is, however, feasible to equip a limited number of laboratories as Centres of Excellence in Africa (CEA), which will receive and process samples from a network of peripheral laboratories in the continent. Cheaper, sensitive and portable real-time PCR methods can be used in peripheral laboratories to pre-screen and select samples for targeted deep sequence or genome wide analyses at these CEAs.

Immunoglobulin G subclass and antibody avidity responses in Malian children immunized with Plasmodium falciparum apical membrane antigen 1 vaccine candidate FMP2.1/AS02A.

BACKGROUND: A malaria vaccine based on Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3, and IgG4) and for antibody avidity. METHODS: Samples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden. RESULTS: AMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90-240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90-240. CONCLUSIONS: Neither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Prevalence of molecular markers of sulfadoxine-pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan.

BACKGROUND: In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan. METHODS: A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing. RESULTS: The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance. CONCLUSION: The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections.

BACKGROUND: Greater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density "asymptomatic" infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions. METHODS: A highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons. RESULTS: The lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards-equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16-22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season. CONCLUSIONS: The DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

Reduced ex vivo susceptibility of Plasmodium falciparum after oral artemether-lumefantrine treatment in Mali.

BACKGROUND: Artemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether-lumefantrine (AL) treatment. METHODS: Study samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing. RESULTS: Overall 61 samples were successfully analysed in ex vivo studies. Mean IC50s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%. CONCLUSION: Malian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

Vaccines against malaria.

Despite global efforts to control malaria, the illness remains a significant public health threat. Currently, there is no licensed vaccine against malaria, but an efficacious vaccine would represent an important public health tool for successful malaria elimination. Malaria vaccine development continues to be hindered by a poor understanding of antimalarial immunity, a lack of an immune correlate of protection, and the genetic diversity of malaria parasites. Current vaccine development efforts largely target Plasmodium falciparum parasites in the pre-erythrocytic and erythrocytic stages, with some research on transmission-blocking vaccines against asexual stages and vaccines against pregnancy-associated malaria. The leading pre-erythrocytic vaccine candidate is RTS,S, and early results of ongoing Phase 3 testing show overall efficacy of 46% against clinical malaria. The next steps for malaria vaccine development will focus on the design of a product that is efficacious against the highly diverse strains of malaria and the identification of a correlate of protection against disease.

A field trial to assess a blood-stage malaria vaccine.

BACKGROUND: Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS: In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS: The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS: On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Molecular basis of allele-specific efficacy of a blood-stage malaria vaccine: vaccine development implications.

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02(A), a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02(A) had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen.

Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens mediate parasite sequestration and host immune evasion. Reactivity to 21 PfEMP1 fragments on a protein microarray was measured in serum samples from Malian children aged 1-6 years and adults. Seroreactivity to PfEMP1 fragments was higher in adults than in children; intracellular conserved fragments were more widely recognized than were extracellular hypervariable fragments. Over a malaria season, children maintained this differential seroreactivity and recognized additional intracellular PfEMP1 fragments. This approach has the potential to identify conserved, seroreactive extracellular PfEMP1 domains critical for protective immunity to malaria.

Positive-unlabeled learning identifies vaccine candidate antigens in the malaria parasite Plasmodium falciparum.

Malaria vaccine development is hampered by extensive antigenic variation and complex life stages of Plasmodium species. Vaccine development has focused on a small number of antigens, many of which were identified without utilizing systematic genome-level approaches. In this study, we implement a machine learning-based reverse vaccinology approach to predict potential new malaria vaccine candidate antigens. We assemble and analyze P. falciparum proteomic, structural, functional, immunological, genomic, and transcriptomic data, and use positive-unlabeled learning to predict potential antigens based on the properties of known antigens and remaining proteins. We prioritize candidate antigens based on model performance on reference antigens with different genetic diversity and quantify the protein properties that contribute most to identifying top candidates. Candidate antigens are characterized by gene essentiality, gene ontology, and gene expression in different life stages to inform future vaccine development. This approach provides a framework for identifying and prioritizing candidate vaccine antigens for a broad range of pathogens.

Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan.

BACKGROUND: Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country. METHODS: In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation. RESULTS: Of the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene. CONCLUSIONS: The prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.

Plasmodium falciparum Development from Gametocyte to Oocyst: Insight from Functional Studies.

Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.

Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Genome-wide SNP analysis of Plasmodium falciparum shows differentiation at drug-resistance-associated loci among malaria transmission settings in southern Mali.

Plasmodium falciparum malaria cases in Africa represent over 90% of the global burden with Mali being amongst the 11 highest burden countries that account for 70% of this annual incidence. The persistence of P. falciparum despite massive global interventions is because of its genetic diversity that drives its ability to adapt to environmental changes, develop resistance to drugs, and evade the host immune system. Knowledge on P. falciparum genetic diversity across populations and intervention landscape is thus critical for the implementation of new strategies to eliminate malaria. This study assessed genetic variation with 12,177 high-quality SNPs from 830 Malian P. falciparum isolates collected between 2007 and 2017 from seven locations. The complexity of infections remained high, varied between sites, and showed a trend toward overall decreasing complexity over the decade. Though there was no significant substructure, allele frequencies varied geographically, partly driven by temporal variance in sampling, particularly for drug resistance and antigen loci. Thirty-two mutations in known drug resistance markers (pfcrt, pfdhps, pfdhfr, pfmdr1, pfmdr2, and pfk13) attained a frequency of at least 2% in the populations. SNPs within and around the major markers of resistance to quinolines (pfmdr1 and pfcrt) and antifolates (pfdhfr and pfdhps) varied temporally and geographically, with strong linkage disequilibrium and signatures of directional selection in the genome. These geo-temporal populations also differentiated at alleles in immune-related loci, including, protein E140, pfsurfin8, pfclag8, and pfceltos, as well as pftrap, which showed signatures of haplotype differentiation between populations. Several regions across the genomes, including five known drug resistance loci, showed signatures of differential positive selection. These results suggest that drugs and immune pressure are dominant selective forces against P. falciparum in Mali, but their effect on the parasite genome varies temporally and spatially. Interventions interacting with these genomic variants need to be routinely evaluated as malaria elimination strategies are implemented.

An In Silico Analysis of Malaria Pre-Erythrocytic-Stage Antigens Interpreting Worldwide Genetic Data to Suggest Vaccine Candidate Variants and Epitopes.

Failure to account for genetic diversity of antigens during vaccine design may lead to vaccine escape. To evaluate the vaccine escape potential of antigens used in vaccines currently in development or clinical testing, we surveyed the genetic diversity, measured population differentiation, and performed in silico prediction and analysis of T-cell epitopes of ten such Plasmodium falciparum pre-erythrocytic-stage antigens using whole-genome sequence data from 1010 field isolates. Of these, 699 were collected in Africa (Burkina Faso, Cameroon, Guinea, Kenya, Malawi, Mali, and Tanzania), 69 in South America (Brazil, Colombia, French Guiana, and Peru), 59 in Oceania (Papua New Guinea), and 183 in Asia (Cambodia, Myanmar, and Thailand). Antigens surveyed include cell-traversal protein for ookinetes and sporozoites, circumsporozoite protein, liver-stage antigens 1 and 3, sporozoite surface proteins P36 and P52, sporozoite asparagine-rich protein-1, sporozoite microneme protein essential for cell traversal-2, and upregulated-in-infectious-sporozoite 3 and 4 proteins. The analyses showed that a limited number of these protein variants, when combined, would be representative of worldwide parasite populations. Moreover, predicted T-cell epitopes were identified that could be further explored for immunogenicity and protective efficacy. Findings can inform the rational design of a multivalent malaria vaccine.